Analysis of Recombinant Chitinase in Crude Protein Extract of Pichia Pastoris

Nur Faidatul Alya, Kalana (2018) Analysis of Recombinant Chitinase in Crude Protein Extract of Pichia Pastoris. [Final Year Project Report] (Unpublished)

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Abstract

Chitinase is an enzyme that can catalyse the hydrolytic degradation of chitin, therefore might aids in inhibiting the growth of fungi, as chitin presence in their cell walls. Intracellular recombinant chitinase crude extract in Pichia pastoris was used in this study to verify this. Endochitinase (Chi III) and exochitinase (Chi II) recombinant in Pichia pastoris were grown in YPD media with I% Glycerol and 0.5% Methanol at final concentration in incubator shaker (30 C) at 250 rpm to induce crude chitinase expression. Empty vector of ppiZa incorporated Pichia pastoris act as the negative control. The control, chitinase II and III were extracted by breaking Pichia pastoris' cell with liquid nitrogen grinding and eluted with Potassium Phosphate Buffer (pH 7). Quantification of protein was done using Bradford assay with standard curve of 2mg/mL BSA and the size of the recombinant crude chitinase was verified using SDS-PAGE. Low amount of crude chitinase obtained and no bands were observed in SDS-PAGE. Three different concentration (10x, 15x and 20x) of chitinase II and III were used to test on their catalytic activity towards fungi. However, there were no inhibition area was formed. For future study, it is recommended to increase the concentration of crude protein and uses Minimal Glycerol Medium+ Histidine and Minimal Methanol+ Histidine media to increase the expression of the crude protein of chitinase.

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